1. INTRODUCTION
2. BACKGROUND OF THE INVENTION
2.1. ROOT DEVELOPMENT
2.2. GENES REGULATING ROOT STRUCTURE
2.3. GEOTROPISM
3. SUMMARY OF THE INVENTION
3.1. DEFINITIONS
4. BRIEF DESCRIPTION OF THE FIGURES
5. DETAILED DESCRIPTION OF THE INVENTION
5.1. SCR GENES
5.1.1. ISOLATION OF SCR GENES
5.1.2. EXPRESSION OF SCR GENE PRODUCTS
5.1.3. ANTIBODIES TO SCR PROTEINS AND POLYPEPTIDES
5.1.4. SCR GENE OR GENE PRODUCTS AS MARKERS FOR QUALITATIVE TRAIT LOCI
5.2. SCR PROMOTERS
5.2.1. CIS-REGULATORY ELEMENTS OF SCR PROMOTERS
5.2.2. SCR PROMOTER-DRIVEN EXPRESSION VECTORS
5.3. PRODUCTION OF TRANSGENIC PLANTS AND PLANT CELLS
5.3.1. TRANSGENIC PLANTS THAT ECTOPICALLY EXPRESS SCR
5.3.2. TRANSGENIC PLANTS THAT SUPPRESS ENDOGENOUS SCR EXPRESSION
5.3.3. TRANSGENIC PLANTS THAT EXPRESS A TRANSGENE CONTROLLED BY THE SCR PROMOTER
5.3.4. SCREENING OF TRANSFORMED PLANTS FOR THOSE HAVING DESIRED ALTERED TRAITS
6. EXAMPLE 1: ARABIDOPSIS SCR GENE
6.1. MATERIALS AND METHODS
6.1.1. PLANT CULTURE
6.1.2. GENETIC ANALYSIS
6.1.3. MAPPING
6.1.4. PHENOTYPIC ANALYSIS
6.1.5. MOLECULAR TECHNIQUES
6.1.6. IN SITU HYBRIDIZATION
6.2. RESULTS
6.2.1. CHARACTERIZATION OF THE SCR PHENOTYPE
6.2.2. CHARACTERIZATION OF CELL IDENTITY IN SCR ROOTS
6.2.3. MOLECULAR CLONING OF THE SCR GENE
6.2.4. THE SCR GENE HAS MOTIFS THAT INDICATE IT IS A TRANSCRIPTION FACTOR
6.2.5. SCR IS A MEMBER OF A NOVEL PROTEIN FAMILY
6.2.6. SCR IS EXPRESSED IN THE CORTEX/ENDODERMAL INITIALS AND IN THE ENDODERMIS
6.3. DISCUSSION
6.3.1. THE SCR GENE REGULATES AN ASYMMETRIC DIVISION REQUIRED FOR ROOT RADIAL ORGANIZATION
6.3.2. SCR INVOLVEMENT IN CELL SPECIFICATION OR CELL DIVISION
6.3.3, A ROLE FOR SCR IN EMBRYONIC DEVELOPMENT
6.3.4. TISSUE-SPECIFIC EXPRESSION OF SCR IS REGULATED AT THE TRANSCRIPTIONAL LEVEL
6.3.5. A NEW FAMILY OF TRANSCRIPTIONAL REGULATORS
7. EXAMPLE 2: ENHANCER TRAP ANALYSIS OF ROOT DEVELOPMENT
7.1. MATERIALS AND METHODS
7.1.1. PLANT GROWTH CONDITIONS:
7.1.2. HISTOLOGY AND GUS STAINING:
7.1.3. CONSTRUCTION OF ENHANCER TRAP LINES:
7.2. RESULTS
7.2.1. DIFFERENTIATION IN THE LRP
7.2.2. MARKER LINES
7.2.3. ET199 PROVIDES EVIDENCE FOR THE ROLE OF SCR IN PLANT DEVELOPMENT
8. EXAMPLE 3: ACTIVITY OF ARABIDOPSIS SCR PROMOTER IN TRANSGENIC ROOTS
9. EXAMPLE 4: ISOLATION SCR SEQUENCES USING PCR-CLONING STRATEGY
10. EXAMPLE 5. EXPRESSION PATTERN OF MAIZE ZCR GENE IN ROOT TISSUE
11. EXAMPLE 6. EXPRESSION PATTERN OF ZCR GENE IN SOYBEAN ROOTS AND ROOT NODULES
12. EXAMPLE 7. SCR EXPRESSION AFFECTS GRAVITROPISM OF AERIAL STRUCTURES
13. DEPOSIT OF MICROORGANISMS
The present invention generally relates to the SCARECROW (SCR) gene family and their promoters. The invention more particularly relates to ectopic expression of members of the SCARECROW gene family in transgenic plants to artificially modify plant structures. The invention also relates to utilization of SCARECROW promoter for tissue and organ specific expression of heterologous gene products.
Asymmetric cell divisions, in which a cell divides to give two daughters with different fates, play an important role in the development of all multicellular organisms. In plants, because there is no cell migration, the regulation of asymmetric cell divisions is of heightened importance in determining organ morphology. In contrast to animal embryogenesis, most plant organs are not formed during embryogenesis. Rather, cells that form the apical meristems are set aside at the shoot and root poles. These reservoirs of stem cells are considered to be the source of all post-embryonic organ development in plants. A fundamental question in developmental biology is how meristems function to generate plant organs.
2.1. ROOT DEVELOPMENT
Root organization is established during embryogenesis. This organization is propagated during postembryonic development by the root meristem. Following germination, the development of the postembryonic root is a continuous process, a series of initials or stem cells continuously divide to perpetuate the pattern established in the embryonic root (steeves and Sussex, 1972, Patterns in Plant Development, Englewood Cliffs, N.J.: Prentice-Hall, Inc.).
Due to the organization of the Arabidopsis root it is possible to follow the fate of cells from the meristem to maturity and identify the progenitors of each cell type (Dolan et al., 1993, Development 119:71-84). The Arabidopsis root is a relatively simple and well characterized organ. The radial organization of the mature tissues in the Arabidopsis root has been likened to tree rings with the epidermis, cortex, endodermis and pericycle forming radially symmetric cell layers that surround the vascular cylinder (FIG. 1A). See also Dolan et al., 1993, Development 119:71-84. These mature tissues are derived from four sets of stem cells or initials: i) the columella root cap initial; ii) the pericycle/vascular initial; iii) the epidermal/lateral root cap initial; and iv) the cortex/endodermal initial (Dolan et al., 1993, Development 119:71-84). It has been shown that these initials undergo asymmetric divisions (Scheres et al., 1995, Development 121:53-62). The cortex/endodermal initial, for example, first divides anticlinally (in a transverse orientation) (FIG. 1B), This asymmetric division produces another initial and a daughter cell. The daughter cell, in turn, expands and then divides periclinally (in the longitudinal orientation) (FIG. 1B). This second asymmetric division produces the progenitors of the endodermis and the cortex cell lineages (FIG. 1B).
2.2. GENES REGULATING ROOT STRUCTURE
Mutations that disrupt the asymmetric divisions of the cortex/endodermal initial have been identified and characterized (Benfey et al., 1993, Development 119:57-70; Scheres et al., 1995, Development 121:53-62). short-root (shr) and scarecrow (scr) mutants are missing a cell layer between the epidermis and the pericycle. In both types of mutants the cortex/endodermal initial divides anticlinally, but the subsequent periclinal division that increases the number of cell layers does not take place (Benfey et al., 1993, Development 119:57-70; Scheres et al., 1995, Development 121:53-62). The defect is first apparent in the embryo and it extends throughout the entire embryonic axis which includes the embryonic root and hypocotyl (Scheres et al., 1995, Development 121:53-62). This is also true for the other radial organization mutants characterized to date, suggesting that radial patterning that occurs during embryonic development may influence the post-embryonic pattern generated by the meristematic initials (Scheres et al., 1995, Development 121:53-62).
Characterization of the mutant cell layer in shr indicated that two endodermal-specific markers were absent (Benfey et al., 1993, Development 119:57-70). This provided evidence that the wild-type SHR gene may be involved in specification of endodermis identity.
2.3. GEOTROPISM
In plants, the capacity for gravitropism has been correlated with the presence of amyloplast sedimentation. See, e.g., Volkmann and Sievers, 1979, Encyclopedia Plant Physiol., N. S. vol 7, pp. 573-600; Sack, 1991, Intern. Rev. Cytol. 127:193-252; Bjxc3x6rkmann, 1992, Adv. Space Res. 12:195-201; Poff et al., in The Physiology of Tropisms, Meyerowitz and Somerville (eds); Cold Spring Harbor Laboratory Press, Plainview, N.Y. (1994) pp. 639-664; Barlow, 1995, Plant Cell Environ. 18:951-962. Amyloplast sedimentation only occurs in cells in specific locations at distinct developmental stages. That is, when and where sedimentation occurs is precisely regulated (Sack, 1991, Intern. Rev. Cytol. 127:193-252). In roots, amyloplast sedimentation only occurs in the central (columella) cells of the rootcap; as these cells mature into peripheral cap cells, the amyloplasts no longer sediment (Sack and Kiss, 1989, Amer. J. Bot. 76:454-464: Sievers and Braun, in The Root Cap: Structure and Function, Wassail et al. (eds.), New York: M. Dekker (1996) pp. 31-49). In stems of many plants, including Arabidopsis, amyloplast sedimentation occurs in the starch sheath (endodermis) especially in elongating regions of the stem (von Guttenberg, Die Physiologischen Scheiden, Handbuch der Pflanzenanatomie; K. Linsbauer (ed.), Berlin: Gebruder Borntraeger, vol. 5 (1943) p. 217; Sack, 1987, Can. J. Bot. 65:1514-1519; Sack, 1991, Intern. Rev. Cytol. 127:193-252; Caspar and Pickard, 1989, Planta 177:185-197; Volkmann et al., 1993, J. Pl. Physiol. 142:710-6).
Gravitropic mutants have been studied for evidence that proves the role of amyloplast sedimentation in gravity sensing. However, many gravitropic mutations affect downstream events such as auxin sensitivity or metabolism (Masson, 1995, BioEssays 17:119-127). Other mutations seem to affect gene products that process information from gravity sensing. For example, the lazy mutants of higher plants and comparable mutants in mosses can clearly sense and respond to gravity, but the mutations reverse the normal polarity of the gravitropic response (Gaiser and Lomax, 1993, Plant Physiol. 102:339-344; Jenkins et al., 1986, Plant Cell Environ 9:637-644). Other mutations appear to affect gravitropism of specific organs. For example, sgr mutants have defective shoot gravitropism (Fukaki et al., 1996, Plant Physiol. 110:933-943; Fukaki et al., 1996, Plant Physiol. 110:945-955; Fukaki et al., 1996, Plant Res. 109:129-137).
Citation or identification of any reference herein shall not be construed as an admission that such reference is available as prior art to the present invention.
The structure and function of a regulatory gene, SCARECROW (SCR), is described. The SCR gene is expressed specifically in root progenitor tissues of embryos, and in certain tissues of roots and stems. SCR expression controls cell division of certain cell types in roots, and affects the organization of root and stem. The invention relates to the SCARECROW (SCR) gene (which encompasses the Arabidopsis SCR gene and its orthologs and paralogs), SCR gene products, (including but not limited to transcriptional products such as mRNAs, antisense and ribozyme molecules, and translational products such as the SCR protein, polypeptides, peptides and fusion proteins related thereto), antibodies to SCR gene products, SCR regulatory regions and the use of the foregoing to improve agronomically valuable plants.
The invention is based, in part, on the discovery, identification and cloning of the gene responsible for the scarecrow phenotype. In contrast to the prevailing view that the SCR gene was likely to be involved in the specification of endodermis, the inventors have determined that the mutant cell layer in roots of scr mutants has differentiated characteristics of both cortex and endodermis. This is consistent with a role for SCR in the regulation of the asymmetric cell division rather than in specification of the identity of either cortex or endodermis. The inventors have also determined that SCR expression affects the gravitropism of plant aerial structures such as the stem.
One aspect of the invention relates to the heterologous expression of SCR genes and related nucleotide sequences, and specifically the Arabidopsis SCR genes, in stably transformed higher plant species. Modulation of SCR expression levels can be used to advantageously modify root and aerial structures of transgenic plants and enhance the agronomic properties of such plants.
Another aspect of the invention relates to the use of promoters of SCR genes, and specifically the use of Arabidopsis SCR promoter to control the expression of protein and RNA products in plants. Plant SCR promoters have a variety of Uses, including but not limited to expressing heterologous genes in the embryo, root, root nodule, and stem of transformed plants.
The invention is illustrated by working examples described infra which demonstrate the isolation of the Arabidopsis SCR gene using insertion mutagenesis. More specifically, T-DNA tagging of genomic and cDNA clones of the Arabidopsis SCR gene are described. Additional working examples include the isolation of SCR sequences from plant genomes using PCR amplification in combination with screening of genomic libraries, and heterologous gene expression in transgenic plants using SCR promoter expression constructs.
Structural analysis of the deduced amino acid sequence of Arabidopsis SCR protein indicates that SCR encodes a transcription factor. Northern analysis, in situ hybridization analysis and enhancer trap analysis show highly localized expression of Arabidopsis SCR in embryos and roots. Genetic analysis shows SCR expression also affects gravitropism of aerial structures (e.g., stems). This indicates that SCR is also expressed in those structures.
Computer analysis of the deduced amino acid sequence of Arabidopsis SCR protein with those of Expressed Sequence Tag (EST) sequences in GenBank reveals the existence of at least thirteen SCR genes in Arabidopsis, one SCR gene in maize, four SCR genes in rice, and one SCR gene in Brassica. A further aspect of the invention relates to the use of such EST sequences to obtain larger and/or complete clones of the corresponding SCR gene.
The various embodiments of the claimed invention presented herein are by the way of illustration and are not meant to limit the invention.
3.1. DEFINITIONS
As used herein, the terms listed below will have the meanings indicated.
35S=cauliflower mosaic virus promoter for the 35S transcript
cDNA=complementary DNA
cis-regulatory element=A promoter sequence 5xe2x80x2 upstream of the TATA box that confers specific regulatory response to a promoter containing such an element. A promoter may contain one or more cis-regulatory elements, each responsible for a particular regulatory response
coding sequence=sequence that encodes a complete or partial gene product (e.g., a complete protein or a fragment thereof)
DNA=deoxyribonucleic acid
EST=expression tagged
functional portion=a functional portion of a promoter is any portion of a promoter that is capable of causing transcription of a linked gene sequence, e.g., a truncated promoter
gene fusion=a gene construct comprising a promoter operably linked to a heterologous gene, wherein said promoter controls the transcription of the heterologous gene
gene product=the RNA or protein encoded by a gene sequence
gene sequence=sequence that encodes a complete gene product (e.g., a complete protein)
GUS=1.3-xcex2-Glucuronidase
gDNA=genomic DNA
heterologous gene=In the context of gene constructs, a heterologous gene means that the gene is linked to a promoter that said gene is not naturally linked to. The heterologous gene may or may not be from the organism contributing said promoter. The heterologous gene may encode messenger RNA (mRNA), antisense RNA or ribozymes
homologous promoter=a native promoter of a gene that selectively hybridizes to the sequence of a SCR gene described herein
mRNA=messenger RNA
operably linked=A linkage between a promoter and gene sequence such that the transcription of said gene sequence is controlled by said promoter
ortholog=related gene in a different plant (e.g., maize ZCARECROW gene is an ortholog of the Arabidopsis SCR gene)
paralog=related gene in the same plant (e.g., Arabidopsis SRPa1 is a paralog of Arabidopsis SCR gene)
RNA=ribonucleic acid
RNase=ribonuclease
SCR=SCARECROW gene or gene product, encompasses (italic) SCR and ZCR genes and their orthologs and paralogs
SCR=SCARECROW protein
scr=scarecrow mutant (e.g., scr1) (lower case)
ZCR=maize ZCARECROW gene, a paralog of, for example, the Arabidopsis SCR gene
SCR protein means a protein containing sequences or a domain substantially similar to one or more motifs (i.e., Motif I-VI), preferably MOTIF III (amino acid residues 373-435 of SEQ ID NO:2) (VHIID) (amino acid residues 8-12 of SEQ ID NO:12), or Arabidopsis SCR protein as shown in FIGS. 13A-F and FIGS. 15A-S. SCR proteins include SCR ortholog and paralog proteins having the structure and activities described herein.
SCR polypeptides and peptides include deleted or truncated forms of the SCR protein, and fragments corresponding to the SCR motifs described herein.
SCR fusion proteins encompass proteins in which the SCR protein or an SCR polypeptide or peptide is fused to a heterologous protein, polypeptide or peptide.
SCR gene, nucleotides or coding sequences means nucleotides, e.g., gDNA or cDNA encoding SCR protein, SCR polypeptides or peptides, or SCR fusion proteins.
SCR gene products include transcriptional products such as mRNAs, antisense and ribozyme molecules, as well as translational products of the SCR nucleotides described herein including but not limited to the SCR protein, polypeptides, peptides and/or SCR fusion proteins.
SCR promoter means the regulatory region native to the SCR gene in a variety of species, which promotes the organ and tissue specific pattern of SCR expression described herein.